The present invention relates to a previously unknown aspartic protease present in human liver, isolated by cloning of a gene from a human liver cDNA library.
This application claims priority to provisional patent application No. 60/031,196 entitled "Napsin, An Aspartic Protease Present in Human Liver" filed Nov. 20, 1996, by Jordan J. N. Tang, Xinli Lin, and Gerald Koelsch, and provisional patent application No. 60/046,126 entitled "Cloning and Gene Structure of Human Napsin" filed May 9, 1997, by by Jordan J. N. Tang, Xinli Lin, and Gerald Koelsch.
Members of the aspartic protease family are characterized by the presence of catalytic aspartic acid residues in their active center. There are five aspartic proteases known to be present in human body. Pepsin and gastricsin are secreted into the stomach for food digestion. Gastricsin is also present in the seminal plasma. Cathepsin D and cathepsin E are present intracellularly to carry out protein catabolism. Renin, which is present in the plasma, is the key enzyme regulating the angiotensin system and ultimately the blood pressure.
Eukaryotic, including human, aspartic proteases are homologous in protein and gene sequences, but have different amino acid and nucleotide sequences. The cDNA and genes of all five human aspartic proteases have been cloned and sequenced. They are synthesized as a single chain zymogen of about 380 residues, which are either secreted or directed to intracellular vacuoles. Upon activation by a self-catalyzed process (except prorenin), an N-terminal pro segment of about 45-residues is cleaved off to produce mature enzymes (Tang and Wong, J. Cell. Biochem. 33, 53-63 (1987)). In some cases, for example, with cathepsin D and renin, mature proteases are further cut into two chains. The three-dimensional structures of the aspartic proteases are very similar. Each enzyme contains two internally homologous lobes (Tang et al., Nature 271, 618-621 (1978)). The active-site cleft, which can accommodate eight substrate residues, and two catalytic aspartic acids, are located between the lobes.
These proteases have distinct and important physiological roles. In addition to their importance in physiological functions, these enzymes are also associated with pathological states. For example, human pepsin and gastricsin are diagnostic indicators for stomach ulcer and cancer (Samloff, Gastroenterology 96, 586-595 (1989); Miki et al., Jpn. J. Cancer Res. 84, 1086-1090 (1993)). Cathepsin D is located in the lysosome. Its main function is the catabolism of tissue proteins. Recent evidence from mice without a functional cathepsin D gene, however, indicates that this enzyme plays a role in the development of intestine in newborn animals. Cathepsin D is also associated with human breast cancer metastasis (Rochefort, Acta Oncologica 31, 125-130 (1992)). Cathepsin E is located in the endoplasmic reticulum of some cells, such as erythrocyte and stomach mucosa cells. It has been applied in the processing of antigens in the immune cells.
Human aspartic proteases have important medical uses. The levels of the proenzymes of human pepsinogen and progastricisin present in the bloodstream and the ratio between the two levels is used in the diagnostic screening of human stomach cancer (Defize, et al., Cancer 59, 952-958 (1987); Miki, et al., Jpn. J. Cancer Res. 84, 1086-1090 (1993)) and ulcer (Miki, et al., Adv. Exp. Med. Biol. 362, 139-143 (1995)). The secretion of procathepsin D is elevated in breast cancer tissue. Thus, the level of procathepsin D in breast cancer is used for clinical prognosis (Rochefort, Acta Oncologica 31, 125-130 (1992)). The analysis of renin in the diagnosis of hypertension is a routine clinical procedure (Brown et al., Handbook of Hypertension 1, 278-323 Robertson, editor (Elsevier Science Publishers, Amsterdam, 1983).
These examples establish that human aspartic proteases are related to human diseases and additional, previously unidentified aspartic proteases, are likely to have clinical applications.
It is therefore an object of the present invention to provide a previously unidentified aspartic protease.
It is a further object of the present invention to characterize and to clone the aspartic protease.
It is still another object of the present invention to identify the tissues in which the aspartic protease is expressed and applications in clinical chemistry and diagnostics.